Over 35 million people worldwide are infected with HIV. Development of an effective HIV vaccine is urgently needed for eradicating the disease. A number of neutralizing antibodies that are broadly effective across numerous HIV strains target the membrane proximal external region (MPER) of the gp41 envelope protein. MPER is a conserved, short and linear epitope which makes it an attractive vaccination target. Unfortunately, vaccines that induce potent, broadly neutralizing antibodies have not yet been developed. A promising subset of MPER vaccine strategies have emerged that involve presenting the MPER in the context of a lipid membrane. To date, these approaches typically use lipopeptides; peptides that are covalently anchored by fatty alkyl tail groups. However, the use of lipopeptides presents drawbacks such as difficulties in peptide purification and propensity for aggregation and membrane-disruption. We recently discovered an enabling method for non-covalent functionalization of preformed liposomes with conventionally synthesized peptides through simple aqueous incubation. When chelated with cobalt (but not other metals), preformed liposomes incorporating cobalt-porphyrin-phospholipid (Co-PoP) potently bind polyhistidine-tagged (his-tagged) peptides. In preliminary results, we have shown this facile approach is effective with conventionally-synthesized his-tagged MPER peptides and Co-PoP liposomes embedded with lipid Toll-like receptor (TLR) ligands. A potent anti-MPER titer was induced in mice. In proof-of-principle studies, we showed that the IgG produced in vaccinated mice inhibited HIV-1 entry in vitro. We propose to explore this platform with MPER peptides for raising neutralizing antibodies with higher titers (Aim 1) and improved neutralization efficacy (Aim 2). In Aim 1, we propose to manipulate this system to increase anti-MPER titers by 1-2 orders of magnitude. We will examine how factors such as MPER density and administration route affect the titer. In unpublished preliminary screening, we found that co-administration of various TLR ligands impacts MPER IgG titers. This will be systematically examined with a panel of TLR ligands. We anticipate developing a broadly-applicable liposomal vaccine strategy to enhance anti-MPER titers. In Aim 2, we will examine the effect of MPER vaccine membrane proximity in affecting neutralization efficacy. The Co-PoP approach enables rapid examination of various conventionally-synthesized peptides since the post-labeling process is trivial. Through assessment of a series MPER peptide fragments of increasingly shorter and more proximal nature, we will assess the optimal MPER sequence to induce neutralizing IgGs for viral entry inhibition. Inhibition will be normalized by the amount of IgG titer to gain a measure of antibody efficacy. Serum, antibodies and (anticipated) monoclonal antibodies will be assessed for HIV-1 neutralization.